The association, reassociation, renaturation or hybridization of complementary nucleic acids (RNA or DNA) in vitro has proven to be a powerful tool for analyzing the genetic material. While nucleic acid reassociation has been used to answer many important questions, two major limitations for its use in many significant biological experiments are the rate and specificity of the association.
Association, reassociation, renaturation, and hybridization are generally used interchangeably to refer to the formation of double-stranded nucleic acids from two single-stranded nucleic acid molecules, whose nucleotide composition allows enough hydrogen-bonds to form between corresponding nucleotides (C-G and A-T or A-U) of these paired single-stranded molecules to prevent the double-stranded molecules from denaturation. The formation of a duplex molecule with all perfectly formed hydrogen-bonds between corresponding nucleotides will be referred as “matched” and duplexes with single or several pairs of nucleotides that do not correspond as “mismatched.” Any combination of single-stranded RNA or DNA molecules can form duplex molecules (DNA:DNA. DNA:RNA, RNA:DNA. or RNA:RNA) under appropriate experimental conditions.
The thermodynamic parameters of association for completely matched nucleic acids are well understood and depend on the nucleotide composition of each pair, their concentration, and the composition of the solution used for these reactions. The nucleotide composition of single-stranded molecules directly influences the temperature of the reaction. Generally, longer molecules, RNA:RNA duplexes, and molecules containing higher G and C nucleotide composition have a higher melting temperature (Tm, the temperature at which 50% of the double-stranded molecules are denatured). In order to achieve the maximum rate, the reactions are usually performed 10-20 degrees Centigrade below the Tm.
Because the kinetics of these reactions are second-order, the rate of the reaction is determined by the concentration of the most abundant species. Low concentrations of the hybridizing species lengthens the time of the reaction. Therefore, the reaction time is one concern when reassociating nucleic acids. It is common to perform the reaction for several hours or even days; however shorter incubation times can be achieved by increasing the quantities of single-stranded nucleic acid molecules, though this is often not desirable.
Finally, the reaction rates depend on the ionic strength of the solution. The single-stranded nucleic acid molecules are negatively charged and thus repel one another; therefore salt should be included for efficient hybridization. The rate varies significantly with decreasing ionic strength below 0.4 M, but is less dependent at higher salt concentrations.
From a theoretical viewpoint, the association of two completely matched single-stranded molecules is well understood. However, in practice, populations of molecules interact resulting in a more complex situation, especially in mixtures of many different single-stranded molecules. A major difficulty in the association of complex mixtures of nucleic acids is the tendency to form duplexes containing one or several mismatched (mispaired, nonspecific) nucleotides in addition to the completely matched duplexes. The degree of discrimination between perfectly matched duplexes and mismatched duplexes is referred to as “specificity.” Generally, duplexes with mismatched nucleotides have a lower Tm than matched ones; however, the magnitude of the decrease depends on many factors such as the duplex length, the position of the mismatched nucleotide pair in the duplex, the type of mismatch (G-A, G-G, G-T, C-C, and etc.), and the neighboring nucleotide composition around the mismatch. To maintain specificity in duplex molecule formation, the association reactions are carried out at temperatures as close as possible to the Tm to prevent formation of mismatched duplexes. Denaturation curves of duplex nucleic acids have a sigmoidal form, and duplexes with different nucleotide sequences but similar Tm's are generally present in the mixture, at least to some extent, at any incubation temperature.
From a practical point of view, shorter single-stranded nucleic acid molecules which have lower Tm's are preferred for a more specific association reaction. With these shorter molecules, even a single nucleotide mismatch can significantly affect the stability of the duplex resulting in a significant decrease in its Tm, though for longer molecules it often does not have such a marked effect. The destabilizing effect of the mismatch is most accentuated at the Tm of the perfectly matched duplex, thus allowing the best discrimination between them to occur at this temperature. There are several different methods for calculating the Tm for short single-stranded nucleic acids (oligos). A generally accepted, common formula is:Tm(° C.)=(number of C's and G's)×4+(number of A's and T's)×2.Thus, for example, for a 20-nucleotide long oligo with equal contents of A, T. G. and C, the Tm of the perfectly matched duplex with a second complementary oligo will be around 60° C.; the difference between this and the single nucleotide mismatched duplex can be as little as 2° C. In such a case, incubation at the Tm will form 50% matched duplex oligos and a considerable fraction of mismatched duplexes. However incubation at temperatures 10-20° C., below the Tm, where the rate is highest, will form both duplexes with high efficiency (95%), making it impossible to distinguish the different species by Tm alone.
Moreover, the Tm for each mismatched duplex is difficult to calculate and can be determined only experimentally. The specificity can be increased by decreasing salt concentration, and the association can be performed even in the absence of salt. But, again, the optimum conditions for specific association can only be determined experimentally for each pair of single-stranded nucleic acids. Moreover, low salt concentration greatly decreases the rate of the reaction.
All of the problems described above make it difficult to achieve both rapid reaction rate and high specificity in association of complex nucleic acid mixtures. And although a number of techniques have been developed that increase the basic reaction rate by a factor of up to 1,000 times, an increase in the rate of the reaction does not necessarily—and normally does not—provide an acceptable level of specificity relative to the basic reference reaction in the single phase system.
The most common technique for accelerating the reaction rate has been to increase the salt concentration up to 10 M (see, e.g., U.S. Pat. No. 5,132,207). The addition of various salts to the hybridization solution can increase the rate of the reaction thousands of times. The effect of acceleration using increased salt concentration is achieved by aggregation or precipitation of nucleic acids, thereby increasing local nucleic acid concentrations. Because the specificity of association is decreased with increasing salt concentration, this method of accelerating association is likely not useful in precise mismatch discrimination; no data has been presented to date that suggests otherwise.
Another approach to accelerating the rate of nucleic acid association is the two-phase phenol aqueous emulsion technique (see, e.g., U.S. Pat. No. 5,132,207). However, it can only be used effectively for acceleration of DNA:DNA duplex molecules; the method is not effective for formation of DNA:RNA or RNA:RNA molecules. The phenol aqueous emulsion technique can accelerate DNA:DNA duplex association greater than about 1000-fold; however, RNA:RNA and RNA:DNA duplex formation is accelerated less than about 100 fold. This technique is very sensitive to phenol concentration and temperature. Moreover, the maximum rate of the reaction is reached at higher salt concentrations. The acceleration again is achieved by “increasing the DNA concentration at the phenol:aqueous interface”, and again a high concentration of salt should decrease discrimination between match and mismatch formation. No experiments were reported that would have determined the specificity of association in this method.
Other prior methods focus only on acceleration of association, but not under conditions that would provide for specificity for detection of, for example, only a few nucleotide differences (e.g., single nucleotide changes). In addition, most work has focused on formation of DNA:DNA or RNA:RNA duplexes; little progress on the acceleration of DNA:RNA or RNA:DNA duplexes has been reported.
For example, Pontius has reported that heterogeneous nuclear ribonucleoprotein A can accelerate the association rate of DNA:DNA duplexes (Pontius et al. (1990) Proc. Natl. Acad. Sci. USA 87:8403-8407; U.S. Pat. No. 5,747,254) and that cationic detergents like cetyltrimethylammonium bromide (CTAB) can also accelerate the association rate of DNA:DNA duplexes (U.S. Pat. No. 5,474,911; Pontius et al. (1991) Proc. Natl. Acad. Sci. USA 88:8237-8241), likely by a similar mechanism. In addition, Pontius reports that even 1 mM CTAB is strongly stabilizing for DNA:DNA helices, even at temperatures well above the melting temperature expected for the double-stranded DNA in the absence of detergent (Pontius et al (1991) Proc. Natl. Acad. Sci. USA 88:8237-8241). See also, Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press, 2001 (ISBN:0-87969-576-5), at 6.62: “Rapid Hybridization Buffers.”
Publications by other researchers have shown that CTAB and other CTAB-like detergents can accelerate formation of RNA:RNA molecules (Nedbal et al., Biochemistry (1997) 36:13552-13557). However, none of these references addressed the specificity issue, and none examined the association of DNA:RNA or RNA:DNA. In short, although CTAB was demonstrated to have accelerating effects upon association of DNA:DNA and RNA:RNA duplexes, there was no demonstration of acceleration of the association of DNA:RNA or RNA:DNA duplexes, nor was there any evidence regarding the specificity of association of any nucleic acid duplexes.
A number of hybridization accelerants of unknown composition are available commercially (such as ULTRAHYB™ and HYBSPEE™, Ambion, Austin, Tex., USA); none are described as increasing simultaneously the rate of association and specificity of association.
Discovery of methods and compositions to accelerate association of RNA:DNA molecules in a manner that provides for high specificity (e.g., no mismatched sequences are reassociated, thus allowing for detection of, for example, single nucleotide differences between two sequences) would greatly advance the molecular genomics field.